Multidimensional electrophoresis, particularly two-dimensional (2D) gel electrophoresis, is a key technology in the analysis of complex samples such as mixtures of proteins. More than ten thousand proteins can be resolved using 2D gel electrophoresis. An example of a gel electrophoresis apparatus using two dimensional electrophoresis is the IPGphor™ system by Amersham Pharmacia Biotech, Piscataway, N.J. Typically, isoelectric focusing (IEF), which separates sample components on the basis of the isolectric point of each component, is used as a first separation dimension. Subsequently, the partially resolved sample from the first dimension, which is generally in the form of a gel strip, is manually attached to a slab gel for the second separation dimension. Such slab gels often separate sample components on the basis of size. Generally, IEF requires about five hours and the slab gel separation requires about six to eight hours to complete.
After the proteins are separated in the slab gel, a dye is used to stain the protein, i.e., at least some of the dye binds to the protein. Unbound dye molecules are washed away using a solvent in a de-staining process. The dye molecules bound to the protein are retained in the gel due to the large binding constant between dye and protein. Then the proteins are detected with a densitometer and digital data is stored in a computer for analysis. The separation and detection procedure and data processing are time intensive, normally requiring about 72 hours to complete. electrophoresis column to analyze the effluent from an HPLC. Such an approach may suffer from significant drift as the electrophoresis column is used to analyze many samples. Additionally, the method requires a tremendous amount of time to analyze the HPLC effluent.
Another example of a multidimensional electrophoresis apparatus is shown in U.S. Pat. No. 6,013,165 to Wiktorowicz, et al. This apparatus includes a first electrophoresis dimension and a second electrophoresis dimension.
Current 2D gel technology includes a number of shortcomings. For example, slab gels allow migrating species travel along curved paths of uncertain length because the migration lanes are not well defined. Migration paths of uncertain length prevent the accurate determination of molecular parameters such as, for example, isoelectric points, molecular identities, mass values, and molecular size, that are estimated from the migration distance. Additionally, errors introduced in slab gel migration complicate data analysis and frustrate automation.
Known techniques have poor sensitivity making it impossible, for example, to quantitatively measure proteins with low natural abundance. Furthermore, the dynamic range of known 2D gel techniques for detecting sample components of different concentrations is limited to about one order of magnitude.